Microcalorimetry of enzyme–substrate binding: yeast phosphoglycerate kinase

Abstract

The binding of substrates and various other anions to yeast phosphoglycerate kinase (PGK) has been studied by titration microcalorimetry. Binding of 3-phosphoglycerate to a single high-affinity site (K≈ 2 × 10⁵ dm³ mol^–1) is entropy driven (ΔG⁰≈–30 kJ mol^–1, ΔH⁰≈–6 kJ mol^–1, ΔS⁰≈+80 J K^–1 mol^–1), with additional heat effects arising from deprotonation of the substrate near neutral pH. This binding is inhibited by a range of other anions (chloride, sulfate, triphosphate, other substrates), probably through competition for the same basic site. Nucleotide substrates (ATP and ADP) appear to bind to two separate sites on the enzyme, one of which is competitive with the phosphoglycerate site and with other anions. Microcalorimetry of non-productive ternary complex for mation (PGK–ADP–3-PG) reflects this multiplicity of sites. Comparative experiments with two site-directed mutants of PGK (His-388 → Gln and Arg-168 → Lys) are also reported.